Supporting InformationBernstein et al. 10.1073/pnas.1118859109SI Materials and MethodsNorthern Blots. Log phase cultures grown in appropriate media at30 °C were harvested, and total RNA was isolated using acidphenol purification followed by DNase treatment using Ambion’sTurbo DNA-free kit. Between 5 and 10 μg total RNA was loadedonto Mops buffered agarose (Ambion) denaturing gel and runfor 2 h at 80 V. RNA was transferred to Hybond N+ membraneand UV cross-linked with 12 J/cm2. Blots were washed with 10mL Sigma Perfect Hyb Plus. Oligonucleotide probes comple-mentary to the region of interest were end-labeled with adeno-sine 5′-triphosphate-32P by T4 polynucleotide kinase. Blots wereincubated in 10 mL Sigma Perfect Hyb Plus with radiolabeledprobe overnight at 57 °C, washed, and visualized by phosphor-imaging. Oligonucleotide sequences of probes are below.

sRNA Northern Blots. RNA was harvested as above. Twenty mi-crograms of total RNA was subjected to Northern analysis asdescribed previously (1).

Dcr1 Protein Purification. A recoded CaDCR1 gene was cloned intoa SUMO-RSF-Duet vector creating an N-[His-Sumo-protease site-Dcr1] construct. Escherichia coli strain BL21(DE3)pLysS wastransformed with this vector, and cultures were grown to an OD of0.5 at 37 °C. Dcr1 expression was induced for 4 h at 37 °C with 1mM isopropyl β-D-thiogalactopyranoside, and cells were collectedand frozen in lysis buffer [10 mM Tris (pH 8.0), 300 mM NaCl, 1mM β-mercaptoethanol, 20 mM imidazole, and 10% glycerol].Cells were lysed by sonication, and lysates were cleared by centri-fugation. Supernatant was passed over Ni-NTA agarose beads, andbound proteins were eluted with elution buffer [10 mM Tris (pH8.0), 300 mM NaCl, 1 mM β-mercaptoethanol, 100 mM imid-azole, and 10% glycerol]. Eluted protein was dialyzed into lysisbuffer, and the His and Sumo tags were cleaved with the SUMOprotease kit (Invitrogen). The cleavage products were passedover Ni-NTA agarose beads, and the flow-through was collected.

Growth Curve: Liquid Growth Curve. Saturated cultures that weregrownovernight inpermissivemediawerediluted toa startingOD600of 0.1. Strains were grown in synthetic complete media at 30 °Ccontaining either glucose ormaltose, andOD600 weremeasured andplotted. Points plotted are the average of three replicates, and errorbars represent 1 SD from the mean of these three replicates.

Solid Media Growth. Saturated overnight cultures were seriallydiluted onto synthetic complete media containing either glucoseor maltose as a carbon source and grown for 2 d at 30 °C.

Plasmids. A recoded CaDCR1 PCR fragment with XbaI/XhoIsites was cloned into p415-TEF (2) to create pV352. pV406 wascreated by cloning a Mung-bean-nuclease-blunted NotI fragmentfrom pAG36 (3) containing the NATR into the HpaI site ofpV352. pV401 was created by cloning a Mung-bean-nuclease-blunted NotI fragment of pAG36 containing NATR into theHpaI site of p415-TEF. The pV397 doxycycline (Dox)-inducibleexpression construct for CaDCR1 in Candida was created byreplacing the SalI/BglII fragment containing GFP from pNIM1(4) with a SalI/BglII PCR fragment containing CaDCR1. Malt-ose-induced CaDCR1 was produced from pV434, which containsa HindIII/PstI PCR fragment of CaDCR1 at the HindIII/PstIsites of pV356. pV356 was created by replacing the HindIII/XhoIflanked CaENO1 promoter from pENO1-EGFP3 (5) with aHindIII/XhoI fragment containing the CaMAL2 promoter. TheNATR-FLP disruption cassette for CaAGO1, pV307, was createdby cloning 1,100 bp immediately upstream of the ORF into theKpnI/ApaI sites of pJK863 (6) and 2,000 bp downstream into theNotI/SacII. pV308, which contains a URA3-FLP disruption cas-sette, was created by cloning the same fragments at the samesites of pSFU1 (7). DAB124 was created by cloning 2,000 bpupstream and downstream of DCR1into JK863 as was for pV307,and DAB126 is DAB124 with the last 500 bp of DCR1 in theplace of the 2,000 bp downstream, and NATR from pJK795;DAB177 is DAB124 with URA3 in place of NATR.

Probe Sequences.

pre-U4 Northern primer: 5′ CTCGGACGAATCCTCACTG-ATACACG

sRNA Northern primer 1: 5′ GATGTCCTGGAAATGGAT-ACCA

sRNA Northern primer 2: 5′ TATCCTGATGATTTCAATA-TCT

sRNA Northern primer 3: 5′ ATTCTTGATCACATCATC-AGCT

pre-rRNA Northern primer: 5′ GCCTATGCTAAACAGAC-CCACC

ACT1 Northern primer: 5′ CCATATCGTCCCAGTTGGAAA

1. Drinnenberg IA, et al. (2009) RNAi in budding yeast. Science 326:544e550.2. Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expres-

sion of heterologous proteins in different genetic backgrounds. Gene 156:119e122.

3. Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes forgene disruption in Saccharomyces cerevisiae. Yeast 15:1541e1553.

4. Park YN, Morschhäuser J (2005) Tetracycline-inducible gene expression and genedeletion in Candida albicans. Eukaryot Cell 4:1328e1342.

5. Staab JF, Bahn YS, Sundstrom P (2003) Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans.Microbiology 149:2977e2986.

6. Shen J, Guo W, Köhler JR (2005) CaNAT1, a heterologous dominant selectable markerfor transformation of Candida albicans and other pathogenic Candida species. InfectImmun 73:1239e1242.

7. Morschhäuser J, Michel S, Staib P (1999) Sequential gene disruption in Candida albicansby FLP-mediated site-specific recombination. Mol Microbiol 32:547e556.

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Fig. S1. Deletion of AGO1 does not impact growth rate. WT [BWP17 (◆)], ago1/AGO1 [VY523 ( )], and ago1/ago1 [VY537 ( )] were grown on a roller drumin synthetic complete (SC) glucose. OD600 was used to measure growth. Points plotted are the average ODs of three replicates, and error bars represent 1 SD ofthe mean.

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Fig. S2. DCR1 deletion impact on Candida albicans growth. (A) CAI4 (◆), DAB157 ( ), DAB224 ( ), and DAB228 ( ) grown on a roller drum in syntheticcomplete (SC) maltose. (B) CAI4 (◆), DAB157 ( ), DAB224 ( ), and DAB228 ( ) grown on a roller drum in SC glucose. (C) BWP17 (◆), DAB151 ( ), DAB184( ), and DAB232 ( ) grown on a roller drum in SC glucose with Dox. (D) BWP17 (◆), DAB151 ( ), DAB184 ( ), and DAB232 ( ) grown on a roller drum in SCglucose. (E) CAI4 (◆), DAB157 ( ), DAB196 ( ), and DAB204 ( ) grown on a roller drum in SC glucose with Dox. (F) CAI4 (◆), DAB157 ( ), DAB196 ( ), andDAB204 ( ) grown on a roller drum in SC glucose. Points plotted are the average ODs of three replicates, and error bars represent 1 SD of the mean. (G) BWP17strains of indicated genotype on SC glucose. (H) Strains from G on SC glucose with Dox. (I) CAI4 strains of indicated genotype on SC glucose. (J) Strains from I onSC glucose with Dox. (K) BWP17 background strains of indicated genotype on SC maltose. (L) Strains as in K on SC glucose. (M) CAI4 strains of indicatedgenotype on SC maltose. (N) strains as in M on SC glucose.

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Fig. S3. Small RNA production in vivo by CaDcr1. (A) Small RNA Northern blot analysis of RNA purified from cells grown in SC maltose or glucose resolved ona denaturing gel. Strains are of indicated genotype, panel is BWP17 background. Blot probed with sRNA Northern primer 2. (B) Northern blot analysis of RNAsamples were purified after being grown in SC glucose or maltose and resolved on a denaturing gel. Strains are of indicated genotype, panel is CAI4 back-ground. Blot probed with sRNA Northern primer 2. Below each blot, ribosomal RNA has been visualized with ethidium bromide to show equal loading. (C andD) Similar to A and B, but a lower-abundance sRNA was probed with sRNA Northern primer 3. (E) Northern blot analysis of sRNA in BWP17 background withsRNA Northern primer 1.

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Fig. S4. DCR1 deletion impact on rRNA processing in Candida albicans. (A–F) Ten micrograms of total RNA was subjected to Northern analysis, probing forunprocessed rRNA using rRNA Northern probe. Strains are of indicated genotype. A, B, E, and F are CAI4 background; C and D are BWP17. Growth condition issynthetic complete with carbon source noted below. Ribosomal RNA sizes are labeled. Blots were stripped and probed for ACT1 transcript as a loading control.(G and H) Northern analysis of U4 processing using pre-U4 Northern probe and ACT1 Northern probe. Strains are of indicated genotype panel in the CAI4background, grown in the condition indicated below. (I) Northern analysis of 10 μg RNA prepared from BWP17 strains of indicated genotype grown in SCglucose probed with pre-rRNA Northern primer or ACT1 northern primer. (J) Northern of 7 μg total RNA from JLK713 grown in glucose (G) or maltose (M) withpre-rRNA Northern primer or ACT1 Northern primer.

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RDN25RDN18

GCCTATGCTAAACAGACCCACC+21 +42

pre-rRNA Northern Primer

Fig. S5. Position of pre-rRNA Northern primer (used in Fig. 4 and Fig. S4) with respect to RDN25 sequence. Numbers indicate distance past the end of RDN25.

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